Although current drug regimens for HIV infection are prolonging millions of lives, CD4+ T lymphocytes harboring latent provirus remain an obstacle to a sterilizing cure. To develop and test clinical interventions, new blood tests are needed that are sensitive enough to detect extremely rare latently-infected cells and accurate enough to measure changes over time. To fully characterize the response to treatment interventions, methods to analyze the properties of single latent cells are also needed. In this project, a micro-fabricated cell culture platform will be used to individually identify and characterize latently-infected cells. Primary lymphocytes will be cultivated in arrays of 105 to over 107 micro-wells, and latent cells will be identified by p24 production after stimulation with anti-CD3/28. After optimization, the sensitivity and reproducibility of this latency assay will be assessed and compared with established assays for HIV DNA and cell-associated infectivity. If the R21 phase succeeds in developing a quantitative assay for replication-competent latent virus, the same platform will be used in the R33 phase to characterize individual latently-infected cells in unprecedented detail, by combining detection based on p24 with measurements of cell-surface phenotype, secreted cytokines, and HIV DNA sequences. Single-cell analyses using these correlated measurements will quantitatively assess the importance of replication-defective proviruses and of homeostatic proliferation.